1. Field of the Invention
The present invention relates to chromogenic and/or fluorogenic mononucleotide-3'-phosphodiesters, and, more particularly, to a novel method for synthesizing such mononucleotide phosphodiesters. These materials may be used, for example, in carrying out various non-isotopic immunoassays.
2. Description of the Prior Art
For a variety of clinical purposes such as, for example, monitoring dosage schedules, monitoring hormone levels, checking for recent ingestion or following pharmacological dynamics of bioavailability, absorption, degradation or excretion, it is a great advantage to measure the concentration of various drugs or the like to the nanomolar or even picomolar level. As is known, radioimmunoassay can accomplish analyses of this type. To carry out an analysis, an acceptable kit or system must include an antiserum, a standard of the compound (i.e., --analyte) to be measured, the radiolabeled derivative of the compound to be measured, a buffering agent or agents and, often, a displacing agent. The antiserum is produced by bleeding animals which have been immunized by innoculation, for example, with the hapten-protein conjugate (immunogen) corresponding to the compound to be measured.
As is well known, in general, the technique of radioimmunoassay measures the competition between radioactively labeled analyte and unlabeled analyte for binding sites on the antibody in the antiserum. By adding to the antiserum known amounts of the analytes to be assayed and a radiolabeled analog, a dose-response curve for bound or free analyte versus concentration of analyte is constructed. After this immunocalibration has been carried out, unknown concentrations can then be compared to the standard dose-response curve for assay. Crucial to this type of assay is the existence of radioactive analytes which compete effectively with non-radioactive analytes. Accordingly, in order to obtain the maximum precision, accuracy, sensitivity, specificity and reproducibility of the assay, purified, well-characterized synthetic radioactive analytes are required.
Several deficiencies in radioimmunoassay methodology have been identified. First of all, it is necessary to make a physical separation of the antibody bound radiolabeled analyte from the free radiolabeled analyte. Further, the methodology is considered rather labor intensive, and the equipment required is likewise relatively expensive, is not uniformly available, and further requires the use of highly trained and skilled technicians to accurately carry out such assays. Likewise, the radioisotopically-labeled analytes are relatively unstable and expensive and pose an increasingly severe waste disposal problem owing to radiation exposure hazards associated with the commonly used radioisotopic labels. Despite these shortcomings, the use of radioimmunoassay has grown considerably.
The substantial recent growth in the use of radioimmunoassay in clinical laboratories has, however, spurred the development of variants which overcome the deficiencies of the radioimmunoassay methodology as described herein. The approaches which have been developed to overcome these deficiencies primarily involve the use of enzyme or fluorescent labels instead of radioisotopic labels, preferably coupled with conditions allowing for measuring a chemical distinction between bound and free fractions of labeled analyte which leads to the elimination of the requirement for physical separation. Immunoassays having the latter simplifing and advantageous feature are referred to as homogeneous immunoassays as opposed to heterogeneous immunoassays where physical separation is required.
Thus, homogeneous immunoassay systems have been developed which are based on the use of an enzyme-labeled analyte where the enzymatic activity of the label is decreased when complexation with the antibody occurs. Unlabeled analyte whose concentration is to be determined displaces the enzyme-labeled analyte bound to the antibody, thus causing an increase in enzymatic activity. Standard displacement or dose-response curves are constructed where increased enzymatic activity (monitored spectophotometrically using what has been termed a "substrate" which ultimately produces a unique chromophore as a consequence of enzyme action) is plotted against increased analyte concentration. These are then used for determining unknown analyte concentrates. The following U.S. patents have been issued in the field of homogeneous enzyme immunoassay: U.S. Pat. Nos. 3,817,837; 3,852,157; 3,875,011; 3,966,556; 3,905,871; 4,065,354; 4,043,872; 4,040,907; 4,039,385; 4,046,636; 4,067,774; 4,191,613; and 4,171,244. In these patents, the label for the analyte is described as an enzyme having a molecular weight substantially greater than 5,000. Also, commercialization of this technology has been limited so far to applications where the analytes are relatively small in molecular size at fluid concentrations of the analyte greater than 10.sup.-10 M.
As a consequence of the limitations of the homogeneous enzyme immunoassay technique described above, considerable effort has been devoted towards developing more sensitive homogeneous immunoassays using fluorescence. These have been primarily directed at assays for the larger sized molecules such as immunoglobulins or polypeptide hormones such as insulin. The following U.S. patents have been issued for this type of assay: U.S. Pat. Nos. 3,998,943; 3,996,345; 4,174,384; 4,161,515; 4,208,479 and 4,160,016. The label in most of these patents involves an aromatic fluorescent molecule, bound either to the analyte or to the antibody. All likewise involve various methods of quenching fluorescence through antibodies or other fluorescent quenchers so that the extent of quenching is related to the amount of analyte present in the sample.
A further type of methodology which may be described as a reactant-labeled fluorescent immunoassay involves the use of a fluorescent-labeled analyte designed so that a fluorescent product is released when it is enzymatically hydrolyzed. Antibody to the analyte portion of the molecule, however, inhibits enzymatic hydrolysis. Consequently, by the law of mass action, fluorescence is enhanced in the presence of increased analyte due to enzymatic hydrolysis of the displaced, fluorescent labeled analyte. As an example, a labeled analyte is .beta.-galactosyl-umbelliferone-sisomicin. The enzyme .beta.-galactosidase cleaves the sugar from the umbelliferone moiety which can then fluoresce. Publications which describe this methodology include: J. F. Burd, R. C. Wong, J. E. Feeney, R. J. Carrico and R. C. Boguolaski, Clin. Chem., 23, 1402(1977); Burd, Carrico, M. C. Fetter, et al., Anal. Biochem., 77, 56 (1977) and F. Kohen, Z. Hollander and Boguolaski, Jour. of Steroid Biochem., 11, 161 (1979).
The previously identified co-pending Farina et al. application provides methodology for carrying out non-isotopic immunoassays which obviates the deficiencies of prior assays of this general type. In an illustrative embodiment, this methodology utilizes a labeled analyte-polypeptide complex which expresses ribonuclease-type activity to catalytically convert a substrate to a chromogenic or fluorogenic reporter molecule.
Many organic compounds have been utilized heretofore for monitoring the catalytic activity of ribonuclease. Such organic compounds, or substrates, as they are commonly referred to, include ribonucleic acid itself, cyclic phosphate diesters, and monoribonucleotide compounds which exhibit the same or similar structural constraints as those expressed by the natural substrate.
Thus, for example, one method for monitoring the catalytic activity of ribonuclease involves the use of a ribonucleic acid solution. That method involves monitoring a decrease in absorbance at 300 nm of a ribonucleic acid solution as a function of time, M. Kunitz, J. Biol. Chem., 164, 563 (1946). Although that method is relatively simple to conduct, it has several deficiencies; specifically, the rate of decrease of absorption is not linear, calibration of each substate solution is required, and direct monitoring of absorbance decreases at 300 nm is impractical with clinical samples.
Another method utilized for monitoring ribonuclease activity is an end-point variant of the procedure described above. In the end point variant procedure, yeast ribonucleic acid is incubated with the enzyme sample for a fixed period of time. The remaining RNA is precipitated with perchloric acid or uranyl acetate/trifluoroacetic acid, and the absorbance of the supernatant is measured after centrifugation. S. B. Anfinsen, R. R. Redfield, W. L. Choate, A. Page, and W. R. Carroll, Jour. Biol. Chem., 207, 201 (1954). However, that method is much too cumbersome for homogeneous immunoassays of the type described in the co-pending Farina et al. application, primarily due to the precipitation step involved.
Yet another variation of the above procedures has been reported by R. C. Kamm, A. G. Smith, and H. Lyons, Analyt. Biochem, 37, 333 (1970). The method described therein is based on the formation of a fluorescent reaction product resulting from the reaction of the dye ethidium bromide with intact yeast ribonucleic acid, but not with the hydrolysis products. In that method, a fluorescent signal, which is monitored, decreases with time. However, monitoring a fluorescent signal which decreases with time is disadvantageous, as the method may result in a lack of sensitivity when only modest differences in enzyme concentration are encountered. In addition, other disadvantages are that the rate of decrease of absorption is not linear, and calibration of each substrate solution is required.
Another known substrate for monitoring ribonuclease activity is a mononucleotide substrate, cytidine 2', 3'-phosphate diester, E. M. Crook, A. P. Mathias, and B. R. Rabin, Biochem. J., 74, 234 (1960). In that method, an increase of absorbance at 286 nm, corresponding to the hydrolysis of the cyclic phosphate ring, is monitored over a two-hour period to measure the ribonuclease activity of the sample. This method, however, cannot be used in homogeneous immunoassay methods of the type described in the Farina et al. co-pending application because there are analyte sample interferences which occur at 286 nm. Furthermore, the distinction between the substrate and product absorbance spectra is small, with the ratio of extinction coefficients being only 1.495 at 286 nm.
Further, certain mononucleotide-3'-phosphodiesters, including, 1-naphthyl esters of 3'-uridylic, 3'-inosonic and 3'-adenylic acids have been utilized as ribonuclease substrates. These napthyl esters have been used to differentiate substrate specificities of ribonucleases from various sources. H. Sierakowska, M. Zan-Kowalczewska, and D. Shugar, Biochem. Biophys. Res. Comm., 19, 138 (1965); M. Zan-Kowalczewska, A. Sierakowska, and D. Shugar, Acta. Biochem. Polon., 13, 237 (1966); H. Sierakowska and D. Shugar, Acta. Biochem. Polon., 18, 143 (1971); H. Sierakowska, H. Szemplinska, D. Shugar, Biochem. Biophys. Res. Comm. 11, 70 (1963). As a result of ribonuclease-induced hydrolysis, the use of such substances results in the liberation of 1-naphthol which is allowed to react with a diazonium salt to form an azo compound having strong visible absorbance. This approach requires that the assay kit include a separately packaged dye former (viz. - a diazonium salt). Also, this substrate cannot be employed in a fluorometric mode.
Various syntheses have been developed heretofore for the preparation of mononucleotide-3'-phosphodiesters. One such method for the preparation of uridine-3'-(1-naphthyl) phosphate is that disclosed in R. Kole and H. Sierakowska, Acta Biochim. Polon, 18, 187 (1971). In accordance with the method shown therein, uridine is acetylated at the 3'-hydroxyl position: ##STR2##
Next the 2'- and 5'-hydroxyl groups of 3'-O-acetyluridine are blocked with dihydropyran; and sequentially the 3'-O-acetyl undergoes hydrolysis so that 2',5'-bis-O-(tetrahydropyranyl) uridine is formed: ##STR3##
Condensation of 2',5'-bis-O-(tetrahydropyranyl)uridine with naphthyl phosphate/dicyclohexylcarbodiimide or naphthyl phosphoryldichloride then results in 1-naphthyl phosphorylation of the 3'-hydroxyl to form the blocked form of the substrate 2',5'-di-O-(tetrahydropyranyl) uridine-3'-(1-naphthyl) phosphate: ##STR4##
The tetrahydropyranyl blocking groups are acid labile and may be removed without competitive phosphate hydrolysis to form the substrate, uridine-3'-(1-naphthyl) phosphate: ##STR5##
A variation of the synthesis described in Sierakowska and Shugar discussed above, is the method described in Rubsamen, Khandler and Witzel (Hoppe-Seyler's) Z. Physiol. Chem., 355, 687 (1974). There, uridine-2',5'-bis-O-(tetrahydropyranyl)-3'-phosphate is prepared by the reaction of dihydropyran with uridine-3'-phosphate. Dephosphorylation of the 2',5'-bis-O-(tetrahydropyranyl)-3'-uridine phosphate with, for example, phosphatase or lead (II) hydroxide, forms 2',5'-di-O-(tetrahydropyranyl) uridine. The 3'-hydroxyl of that compound may then be phosphorylated in the fashion disclosed in Sierakowska and Shugar to form the desired mononucleotide-3'-phosphodiester, such as, for example, uridine-3'-(1-naphthyl) phosphate.
The synthesis schemes described by Sierakowska et al., and Rubsamen et al., suffer, however, from several major deficiencies. For example, in each synthesis method, the preparation of the key intermediate, 2',5'-bis-O-(tetrahydropyranyl)-uridine, involves an undesirable, lengthy chromotagraphy. Further, the resulting product is a mixture of diastereomeric pairs in low yields; and this complicates subsequent synthetic steps. Finally, the overall synthesis is labor-intensive.
Closely similar schemes to those of Sierakowska et al. and Rubsamen et al. are disclosed in Polish Pat. No. 81969. In one synthesis described therein, 2',5'-di-O-tetrahydropyranyl-3'-uridine-(1-naphthyl) phosphate is formed in dicyclohexylcarbodiimide and pyridine by the reaction of a salt of 1-naphthylphosphoric acid, (e.g., the pyridine, aniline, lutidine or tri-n-buytlamine salt of the acid) with 2',5'-di-O-(tetrahydropyranyl)-uridine. In another synthesis described therein, uridine 2'-O-tetrahydropyranyl-5'-O-methyl-3'-(1-naphthyl) phosphate is formed in pyridine by the reaction of a salt of 1-naphthylphosphoric acid and 5'-O-methyl-2'-O-(tetrahydropyranyl)-uridine. These schemes likewise suffer from the deficiencies of the Sierakowska et al. and Rubsamen et al. methods.
In addition, methods are known for preparing oligoribonucleotides which incorporate the synthesis of 2',5'-diblocked nucleotides as intermediates. Thus, in J. Smrt and F. Sorm, Collection Czechoslav. Chem. Commun. 27, 73 (1962), uridylic acid is converted into 5'-O-acetyluridine 2',3'-cyclic phosphate which, after enzymatic cleavage of the cyclic phosphate by pancreatic ribonuclease, results in 5'-O-acetyluridine-3'-phosphate, which is then transformed into 2'-O-tetrahydropyranyl 5'-O-acetyluridine 3'-phosphate by the reaction with dihydropyran.
In this method, acetylation at the 5'-hydroxyl of the cyclic phosphate is utilized as a synthetic convenience for preparing intermediates in the synthesis of oligoribonucleotides. Deblocking of the 5'-acetyl is ultimately carried out in the formation of the desired oligoribonucleotide. This, however, does not describe a suitable method for synthesizing a chromogenic and/or fluorogenic mononucleotide-3'-phosphodiester. Moreover, insofar as is known, the Smrt et al. methods have not heretofore been utilized in making such chromogenic and/or fluorogenic mononucleotide-3'-phosphodiesters, despite the deficiencies of prior methods.
Thus, despite the considerable number of methods that have been developed and utilized for synthesizing various substrates suitable for use for monitoring enzymatic or catalytic activity, there remains the need for further development which can overcome the various shortcomings of the presently known synthetic methods. None of the synthesis schemes described above are currently being used commercially for the manufacture of mononucleotide-3'-phosphodiesters insofar as is known.
It is, accordingly, an object of the present invention to provide a novel method for synthesizing mononucleotide 3'-phosphodiesters having a chromogenic and/or fluorogenic functional group at the 3'-phosphate moiety of the furanoside ring. A related object is to provide a method for synthesizing such mononucleotides in a manner so as to eliminate the formation of undesirable diastereomeric pairs.
Another object is to provide a novel method for synthesizing chromogenic and/or fluorogenic mononucleotide 3'-phosphodiesters which is less labor intensive than previous syntheses.
Yet another object of this invention is to provide a novel synthesis of chromogenic and/or fluorogenic monucleotide 3'-phosphodiesters which results in improved overall yields.
Still another object of the present invention is to provide a novel synthesis of chromogenic and/or fluorogenic mononucleotide 3'-phosphodiesters, which may be carried out on a multigram scale sufficient for commercial use.
These and other objects and advantages of the present invention will become apparent from the following detailed description.
While the invention is susceptible to various modifications and alternative forms, there will herein be described in detail the preferred embodiments. It is to be understood, however, that it is not intended to limit the invention to the specific forms disclosed. On the contrary, it is intended to cover all modifications and alternative forms falling within the spirit and scope of the invention as expressed in the appended claims. For example, while the present invention will be primarily described in conjunction with the formation of a uridine-3'-phosphodiester, it should be appreciated that bases other than uracil may be employed, as will be described herein.